Nasal bots carry relevant titers of CWD prions in naturally infected white-tailed deer.

Chronic wasting disease (CWD) is a prion disease affecting farmed and free-ranging cervids. CWD is rapidly expanding across North America and its mechanisms of transmission are not completely understood. Considering that cervids are commonly afflicted by nasal bot flies, we tested the potential of these parasites to transmit CWD. Parasites collected from naturally infected white-tailed deer were evaluated for their prion content using the protein misfolding cyclic amplification (PMCA) technology and bioassays. Here, we describe PMCA seeding activity in nasal bot larvae collected from naturally infected, nonclinical deer. These parasites efficiently infect CWD-susceptible mice in ways suggestive of high infectivity titers. To further mimic environmental transmission, bot larvae homogenates were mixed with soils, and plants were grown on them. We show that both soils and plants exposed to CWD-infected bot homogenates displayed seeding activity by PMCA. This is the first report describing prion infectivity in a naturally occurring deer parasite. Our data also demonstrate that CWD prions contained in nasal bots interact with environmental components and may be relevant for disease transmission.

Characterization of the complete mitochondrial genomes of the zoonotic parasites Bolbosoma nipponicum and Corynosoma villosum (Acanthocephala: Polymorphida) and the molecular phylogeny of the order Polymorphida.

Acanthocephalans of the order Polymorphida mainly parasitic in birds and mammals, are of veterinary, medical and economic importance. However, the evolutionary relationships of its 3 families (Centrorhynchidae, Polymorphidae and Plagiorhynchidae) remain under debate. Additionally, some species of Polymorphida (i.e. Bolbosoma spp. and Corynosoma spp.) are recognized as zoonotic parasites, associated with human acanthocephaliasis, but the mitochondrial genomes for representatives of Bolbosoma and Corynosoma have not been reported so far. In the present study, the complete mitochondrial genomes B. nipponicum and C. villosum (Acanthocephala: Polymorphidae) are reported for the first time, which are 14 296 and 14 241 bp in length, respectively, and both contain 36 genes [including 12 PCGs, 22 tRNA genes and 2 rRNA genes] and 2 non-coding regions (NCR1 and NCR2). The gene arrangement of some tRNAs in the mitogenomes of B. nipponicum and C. villosum differs from that found in all other acanthocephalans, except Polymorphus minutus. Phylogenetic results based on concatenated amino acid (AA) sequences of the 12 protein-coding genes (PCGs) strongly supported that the family Polymorphidae is a sister to the Centrorhynchidae rather than the Plagiorhynchidae, and also confirmed the sister relationship of the genera Bolbosoma and Corynosoma in the Polymorphidae based on the mitogenomic data for the first time. Our present findings further clarified the phylogenetic relationships of the 3 families Plagiorhynchidae, Centrorhynchidae and Polymorphidae, enriched the mitogenome data of the phylum Acanthocephala (especially the order Polymorphida), and provided the resource of genetic data for diagnosing these 2 pathogenic parasites of human acanthocephaliasis.

Review of metazoan parasites of the northern fur seal (Callorhinus ursinus) and the analysis of the gastrointestinal helminth community of the population on St. Paul Island, Alaska.

The northern fur seal (NFS), Callorhinus ursinus (Mammalia: Otariidae), is a marine mammal species included into the IUCN Red List as the vulnerable species which population is dramatically declining. A significant amount of parasitological data collected previously and our recent data allowed us to clarify the list of NFS metazoan parasites and to perform a comprehensive analysis of the gastrointestinal helminth community. Gastrointestinal tracts from 756 NFSs (3- to 4-year-old males) were collected during the annual Aleut subsistence harvests in July-August of 2011-2014 from five separate rookeries on St. Paul Island, Alaska. Totally, 27,625 specimens of helminths and approximately 1000 nasal mites were collected and identified. Detailed analysis of the previously published and newly obtained data revealed 32 species of metazoan parasites, including trematodes (6 species), cestodes (4), nematodes (9), acanthocephalans (9) and arthropods (4). The gastrointestinal helminth community of newly studied NFSs comprised 19 species including trematodes (4), cestodes (3), nematodes (5) and acanthocephalans (7). Temporal changes in the helminth community structure were small but statistically significant. Gastrointestinal helminth infracommunities comprised from 1 to 10 species (average of 4). Small but significant correlation was found between the abundances of acanthocephalans (Corynosoma similis and C. strumosum), nematodes (Contracaecum osculatum, Pseudoterranova spp.) and cestode Diphyllobothrium tetrapterum.

Multifocal Necrotizing Myopathy in Northern Elephant Seal ( Mirounga angustirostris) Pups, San Miguel Island, California.

A field study addressing causes of mortality in freshly dead northern elephant seals ( Mirounga angustirostris, Gill, 1866) was conducted on San Miguel Island, California, in February 2015. Necropsies were performed on 18 pups ranging in age from stillbirths to approximately 7 to 8 weeks. The primary gross diagnoses in these pups included trauma, myopathy, starvation/emaciation, infections, congenital anomalies, and perinatal mortality. However, 6 (33%) had a previously unrecognized myopathy characterized by multiple white streaks that were most obvious within the inner layer of the abdominal wall and the small innermost ventral intercostal muscles. Following histological examination, 2 more pups from San Miguel Island and 6 pups from The Marine Mammal Center (Sausalito, California) were found to have similar lesions. Histologically, the lesions within the skeletal muscles were characterized by a multifocal polyphasic, mild to severe, acute to subacute necrotizing myopathy with mineralization. Acute necrosis and degeneration characterized by pyknotic nuclei, eosinophilic cytoplasm and cytoplasmic vacuolization were found in smooth muscle myocytes within the urinary bladder and digestive system. Degeneration of myocytes was present in the tunica media of a few small- to medium-sized vessels and was characterized by a vacuolar degeneration and occasionally necrosis. This condition has been termed multifocal necrotizing myopathy. A cause of this myopathy was not identified.

Morphological and molecular evidence for synonymy of Corynosoma obtuscens Lincicome, 1943 with Corynosoma australe Johnston, 1937 (Acanthocephala: Polymorphidae).

Corynosoma obtuscens Lincicome, 1943 (Acanthocephala: Polymorphidae) is synonymised with Corynosoma australe Johnston, 1937 based on combined morphological and molecular evidence. Morphological comparison of C. obtuscens (24 males and 27 females) collected from a California sea lion Zalophus californianus (Lesson) in California, USA, with the type-specimens of C. obtuscens and C. australe, and with published data on C. australe collected from different hosts and regions showed no significant differences. The levels of genetic divergence in the cox1 sequences obtained from C. obtuscens from a California sea lion in the present study and C. australe from otariid seals from Argentina and penguins from Brazil ranged between 1.4-1.6% and was considered to represent intraspecific variability. Additionally, cox1 sequences were generated for Andracantha phalacrocoracis (Yamaguti, 1939), Corynosoma semerme (Forssell, 1904), C. strumosum (Rudolphi, 1802), C. validum Van Cleave, 1953 and C. villosum Van Cleave, 1953. Our results revealed inconsistency in the identification of material used as a source of the previously published sequence data for C. obtuscens and C. magdaleni Montreuil, 1958.

Novel Type of Chronic Wasting Disease Detected in Moose (Alces alces), Norway.

Chronic wasting disease (CWD) persists in cervid populations of North America and in 2016 was detected for the first time in Europe in a wild reindeer in Norway. We report the detection of CWD in 3 moose (Alces alces) in Norway, identified through a large scale surveillance program. The cases occurred in 13-14-year-old female moose, and we detected an abnormal form of prion protein (PrPSc) in the brain but not in lymphoid tissues. Immunohistochemistry revealed that the moose shared the same neuropathologic phenotype, characterized by mostly intraneuronal deposition of PrPSc. This pattern differed from that observed in reindeer and has not been previously reported in CWD-infected cervids. Moreover, Western blot revealed a PrPSc type distinguishable from previous CWD cases and from known ruminant prion diseases in Europe, with the possible exception of sheep CH1641. These findings suggest that these cases in moose represent a novel type of CWD.

Brucella pinnipedialis in lungworms Parafilaroides sp. and Pacific harbor seals Phoca vitulina richardsi: proposed pathogenesis.

Brucella spp. were first isolated from marine mammals in 1994 and since have been described in numerous pinniped and cetacean species with nearly global distribution. Microscopic, electron microscopic, or culture results have shown lungworms in harbor seals to be infected with brucellae, suggesting that the lungworms may serve a role in this infection. In this study, we reviewed archived and more recent case material from 5 Pacific harbor seals from Washington State (USA) with evidence of B. pinnipedialis infection in the lungworm Parafilaroides sp. Twenty-two sections of lung containing approximately 220 Parafilaroides sp., stained with an immunohistochemical technique using antibody to B. abortus, showed approximately 80 (36%) infected nematodes. A few brucellae were also present in lung parenchyma in proximity to nematodes. Infection was present in the first- and fourth-stage larvae in the seal lung and intestines, as well as in the male and female reproductive organs of adult nematodes. Infected sperm deposits in the nematode uterus were suggestive of venereal transmission between lungworms. Massive infection of some degenerate adult lungworms and evidence of degeneration of some developing larvae in utero were observed. Based on these observations, we suggest that Parafilaroides sp., rather than the Pacific harbor seal Phoca vitulina richardsi, is the preferred host of B. pinnipedialis infection.

Tonsil biopsy to detect chronic wasting disease in white-tailed deer (Odocoileus virginianus) by immunohistochemistry.

Chronic wasting disease (CWD) continues to spread in wild and farmed cervid populations. Early antemortem CWD testing of farmed cervids is of considerable interest to producers and regulatory agencies as a tool to combat this spread. The tissues accessible for antemortem sampling are limited and include biopsy of the tonsil and recto-anal mucosa-associated lymphoid tissue (RAMALT). The sensitivity to detect CWD by immunohistochemistry (IHC)-the regulatory gold standard-using biopsy samples of RAMALT from naturally infected white-tailed deer (WTD) has been determined by several studies. However, similar information is lacking for tonsil biopsy. In this study, two-bite tonsil biopsies from 79 naturally infected farmed WTD were used to determine the diagnostic sensitivity of tonsil IHC compared to the official CWD status based on results from the medial retropharyngeal lymph nodes and obex. IHC detection of CWD by tonsil biopsy was compared to the result and follicle metrics from the contralateral whole tonsil. The sensitivity of two-bite tonsil biopsy for detecting CWD by IHC was 72% overall. When the stage of infection was considered, the sensitivity was 92% for deer in late preclinical infection but only 55% for early preclinical infection. For deer with early preclinical infection, the sensitivity for deer homozygous for the prion protein gene (PRNP) coding for glycine at codon 96 (GG) was 66% but only 30% when heterozygous for the serine substitution (GS). The results indicate that the sensitivity of two-bite tonsil biopsy in WTD, and consequently its potential utility as an antemortem diagnostic, is limited during early infection, especially in WTD heterozygous for the serine substitution at PRNP codon 96.

Transmissibility of caprine scrapie in ovine transgenic mice.

BACKGROUND: The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats. RESULTS: First passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports. CONCLUSIONS: These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program.

Update on the prevalence of the hookworm, Uncinaria lucasi, in northern fur seals (Callorhinus ursinus) on St. Paul Island, Alaska, 2011.

Prevalence of hookworms (Uncinaria lucasi Stiles, 1901) was determined in the northern fur seal (Callorhinus ursinus Linnaeus, 1758) on St. Paul Island (SPI), Alaska in July and August, 2011. Three of 61 (4.9%) dead pups harbored 1 to 13 adult hookworms each in their intestines. Parasitic larvae (L(3)) of hookworms were recovered from the blubber of 4 of 133 (3%) of subadult males (SAMs) examined. One parasitic L(3) was detected from each infected SAM. Adult U. lucasi (n = 3) were found in the intestine of 1 of 105 SAMs examined (0.95%). This is the first documented finding of adult U. lucasi in SAMs of the northern fur seals. Continued low prevalence of hookworms the last several years parallels the tremendous decline in the number of fur seals on SPI over a similar time period.

Morphology, molecular characterization and phylogeny of Bolbosoma nipponicum Yamaguti, 1939 (Acanthocephala: Polymorphidae), a potential zoonotic parasite of human acanthocephaliasis.

Human acanthocephaliasis is a rare parasitic zoonosis mainly caused by acanthocephalans belonging to the genera Acanthocephalus, Bolbosoma, Corynosoma, Macracanthorhynchus, and Moniliformis. In the present paper, the juveniles of Bolbosoma nipponicum Yamaguti, 1939 collected from the northern fur seal Callorhinus ursinus (Linnaeus) (Mammalia: Carnivora) in Alaska, USA were precisely identified based on morphological characters and genetic data. Their detailed morphology was studied using light and, for the first time, scanning electron microscopy. The molecular characterization of the nuclear genes [small ribosomal subunit (18S) and large ribosomal subunit (28S)] and the mitochondrial cytochrome c oxidase subunit 1 (cox1) sequence data of B. nipponicum are provided for the first time. Moreover, in order to clarify the phylogenetic relationships of the genus Bolbosoma and the other genera in the family Polymorphidae, phylogenetic analyses were performed integrating different nuclear (18S + ITS+28S) and mitochondrial (cox1) sequence data using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic results showed that Bolbosoma has a sister relationship with Corynosoma, and also revealed that Southwellina is sister to Ibirhynchus + Hexaglandula. Our molecular phylogeny also indicated a possible host-switch pattern during the evolution of the polymorphid acanthocephalans. The ancestors of polymorphid acanthocephalans seem to have originally parasitized fish-eating waterfowl in continental habitats, then extended to fish-eating marine birds in brackish water and marine habitats, and finally, opportunistically infected the marine mammals.

Immunogenicity, Safety, and Anti-Viral Efficacy of a Subunit SARS-CoV-2 Vaccine Candidate in Captive Black-Footed Ferrets (Mustela nigripes) and Their Susceptibility to Viral Challenge.

A preliminary vaccination trial against the emergent pathogen, SARS-CoV-2, was completed in captive black-footed ferrets (Mustela nigripes; BFF) to assess safety, immunogenicity, and anti-viral efficacy. Vaccination and boosting of 15 BFF with purified SARS-CoV-2 S1 subunit protein produced a nearly 150-fold increase in mean antibody titers compared to pre-vaccination titers. Serum antibody responses were highest in young animals, but in all vaccinees, antibody response declined rapidly. Anti-viral activity from vaccinated and unvaccinated BFF was determined in vitro, as well as in vivo with a passive serum transfer study in mice. Transgenic mice that received BFF serum transfers and were subsequently challenged with SARS-CoV-2 had lung viral loads that negatively correlated (p < 0.05) with the BFF serum titer received. Lastly, an experimental challenge study in a small group of BFF was completed to test susceptibility to SARS-CoV-2. Despite viral replication and shedding in the upper respiratory tract for up to 7 days post-challenge, no clinical disease was observed in either vaccinated or naive animals. The lack of morbidity or mortality observed indicates SARS-CoV-2 is unlikely to affect wild BFF populations, but infected captive animals pose a potential risk, albeit low, for humans and other animals.

Antemortem diagnosis of a ventricular septal defect in a California sea lion Zalophus californianus.

A yearling California sea lion Zalophus californianus stranded in poor body condition, and on physical examination a heart murmur was audible bilaterally. The sea lion was diagnosed with a left-to-right shunting membranous ventricular septal defect (VSD) using B-mode, color-flow Doppler and continuous-wave Doppler echocardiography. A left-to-right intracardiac shunting lesion was confirmed during cardiac angiographic computed tomography. The VSD defect was verified during the necropsy examination. On histologic examination concurrent mild multifocal myocarditis with focal mild ventricular free-wall myocardial necrosis were identified. A specific cause for the myocarditis and myocardial necrosis was not found, and association with the VSD and resultant myocardial dysfunction was presumed. This is the first report of the antemortem diagnosis of a VSD in a marine mammal and the first report of a VSD in a California sea lion.

Review of research on hookworms (Uncinaria lucasi Stiles, 1901) in northern fur seals (Callorhinus ursinus Linnaeus, 1758).

The objective of this article is to review knowledge on the hookworm Uncinaria lucasi Stiles, 1901 in northern fur seals, Callorhinus ursinus Linnaeus, 1758. Emphasis is placed on research on this host-parasite system in the Pribilof Islands, AK, USA where the bulk of the studies has been performed.

OPPORTUNISTIC SURVEILLANCE OF CAPTIVE AND FREE-RANGING BIGHORN SHEEP (OVIS CANADENSIS) IN COLORADO, USA, FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES.

Bighorn sheep (Ovis canadensis) are predicted to have a degree of susceptibility to the transmissible spongiform encephalopathies (TSE) chronic wasting disease and scrapie. We opportunistically screened 127 captive bighorn sheep and 152 free-ranging bighorn sheep in Colorado, US for the presence of TSE over a period of 35 yr. None of the animals demonstrated clinical signs, gross pathology, histopathology, or immunohistochemical staining patterns suggestive of TSE.

Regional and Distant Metastasis of Oral Squamous Cell Carcinoma in a Male Castrated Llama: A Case Report.

A 19-year-old male castrated llama presented with a 3-year history of tooth root abscesses and an osseous mass on the left mandible. Surgical excision of the affected teeth and mass was performed, and histopathologic review indicated that the mass was an oral squamous cell carcinoma. The patient was admitted to the hospital again 4 months later in severe respiratory distress with submandibular edema, and bicavitary effusion. Necropsy results revealed regional and distant metastatic squamous cell carcinoma.

Detection of two dissimilar chronic wasting disease isolates in two captive Rocky Mountain elk (Cervus canadensis) herds.

Chronic wasting disease (CWD) continues to spread in both wild and captive cervid herds in North America and has now been identified in wild reindeer and moose in Norway, Finland and Sweden. There is limited knowledge about the variety and characteristics of isolates or strains of CWD that exist in the landscape and their implications on wild and captive cervid herds. In this study, we evaluated brain samples from two captive elk herds that had differing prevalence, history and timelines of CWD incidence. Site 1 had a 16-year history of CWD with a consistently low prevalence between 5% and 10%. Twelve of fourteen naïve animals placed on the site remained CWD negative after 5 years of residence. Site 2 herd had a nearly 40-year known history of CWD with long-term environmental accrual of prion leading to nearly 100% of naïve animals developing clinical CWD within two to 12 years. Obex samples of several elk from each site were compared for CWD prion strain deposition, genotype in prion protein gene codon 132, and conformational stability of CWD prions. CWD prions in the obex from site 2 had a lower conformational stability than those from site 1, which was independent of prnp genotype at codon 132. These findings suggest the existence of different CWD isolates between the two sites and suggest potential differential disease attack rates for different CWD strains.

Experimental oral transmission of chronic wasting disease to red deer (Cervus elaphus elaphus): early detection and late stage distribution of protease-resistant prion protein.

Chronic wasting disease (CWD), an important emerging prion disease of cervids, is readily transmitted by intracerebral or oral inoculation from deer-to-deer and elk-to-elk, suggesting the latter is a natural route of exposure. Studies of host range susceptibility to oral infection, particularly of those species found in habitats where CWD currently exists are imperative. This report describes the experimental transmission of CWD to red deer following oral inoculation with infectious CWD material of elk origin. At 18 to 20 months post-inoculation, mild to moderate neurological signs and weight loss were observed and animals were euthanized and tested using 3 conventional immunological assays. The data indicate that red deer are susceptible to oral challenge and that tissues currently used for CWD diagnosis show strong abnormal prion (PrP(CWD)) accumulation. Widespread peripheral PrP(CWD) deposition involves lymphoreticular tissues, endocrine tissues, and cardiac muscle and suggests a potential source of prion infectivity, a means of horizontal transmission and carrier state.

Epizootic hemorrhagic disease outbreak in a captive facility housing white-tailed deer (Odocoileus virginianus), bison (Bison bison), elk (Cervus elaphus), cattle (Bos taurus), and goats (Capra hircus) in Colorado, U.S.A.

An ungulate research facility in Fort Collins, Colorado, U.S.A., experienced mortality in white-tailed deer (Odocoileus virginianus) because of epizootic hemorrhagic disease virus (EHDV) infection from 20 August 2007 through 26 September 2007. Epizootic hemorrhagic disease virus (EHDV) was detected by reverse transcriptase polymerase chain reaction and virus isolation from the spleen and lung tissues of two white-tailed deer. Virus neutralization tests were performed on pre- and postoutbreak sera from other species maintained in the same facility, including bison (Bison bison), elk (Cervus elaphus), domestic cattle (Bos taurus), and domestic goats (Capra hircus), as well as postoutbreak sera from the surviving white-tailed deer. Serum samples that represented all species in the facility neutralized EHDV-1 and EHDV-2 either before or after the outbreak. The animals that neutralized EHDV-1 did not neutralize EHDV-2. No clinical signs attributable to EHDV infection were noted in any of the species other than the deer during the outbreak. Although experimental EHDV infections have been reported in bison and elk, natural exposures have not been previously documented in these species in North America. The roles that elk, bison, cattle, and goats might play in the epidemiology of EHDV in a close-contact multispecies situation remain unknown.

Causes of mortality in northern elephant seal pups on San Miguel Island, California.

In February 2015, we conducted a field study of causes of mortality of northern elephant seal (Mirounga angustirostris) pups on San Miguel Island, California. Autopsies were performed on 18 freshly dead pups. Ages of pups ranged from stillborn to 6-8 wk. Gross and histologic lesions included trauma (9 of 18 pups), multifocal necrotizing myopathy (8 of 18), starvation with emaciation (7 of 18), congenital anomalies (3 of 18), bacterial infections (3 of 18), and perinatal mortality (stillbirths and neonates; 2 of 18). Trauma and emaciation or starvation were the most significant contributors to death. Bacterial infections included hemolytic Escherichia coli isolated from the lungs of 2 pups with pneumonia. Additionally, non-hemolytic Streptococcus sp. and hemolytic E. coli were isolated from the liver of an emaciated pup that had mild multifocal suppurative hepatitis. Other lesions, including a previously described necrotizing myopathy, congenital anomalies, and bacterial infections, were detected concurrently in cases with starvation and/or emaciation or trauma.